The recent identification and characterization of cDNA clones encoding several different mammalian PDEs has supported the cumulative biochemical evidence for the existence of multiple isozyme families as well as the number and tissue distribution of particular subtypes (Livi, G. P. et al., Mol. Cell. Bio. 10:2678-86 (1990); Colicelli, J. et al., Proc. Natl, Acad. Sci. (USA) 86:3599-3603 (1989) and Davis et al., Proc. Natl, Acad. Acad. (USA) 86:3604-08 (1989)). A particularly interesting isozyme family with respect to drug discovery is the PDE IV family. There is considerable evidence to suggest that this isozyme family represents a molecular target for a variety of therapeutic agents ranging from anti-depressants (Nicholson, C. D. et al., Trends in Pharm. Sci. 12:14-27(1991)) to anti-asthmatic and anti-inflammatory agents (Torphy, T. J. and B. J. Undem, Thorax 46:512-23 (1991)). The cloning, expression and biochemical characteristics of hPDE IV.sub.A, an enzyme encoded by a cDNA obtained from a human monocyte library has been reported (Livi, G. P. et al. supra (1990) and Torphy T. J. et al., J. Biol. Chem. 267:1798-1804 (1992)). The purpose of this invention is to provide cloned and characterized PDE IV subtypes expressed in human brain.
Cyclic nucleotide phosphodiesterases (PDEs) consist of a family of enzymes that catalyze the hydrolysis of 3',5'-cyclic nucleotides, resulting in the formation of 5'-nucleotide metabolites. At least five distinct mammalian PDE isozyme families exist, each distinguished on the basis of a number of biochemical properties including 1) enzyme kinetics, 2) substrate selectivity, and 3) selective inhibition by various compounds. These isozyme families are defined as: I) the Ca.sup.2+ /calmodulin-dependent PDEs; II) the cGMP-stimulated PDEs; III) the cGMP-inhibited PDEs; IV) the cAMP-specific PDEs and V) the cGMP-specific PDEs (Beavo, J. A. et al., Trends Pharmacol. Sci. 11:150-155 (1990) and Conti, M. et al., Endocrine Rev. 12:218-234 (1991)).
There is considerable interest in evaluating inhibitors of the low-K.sub.m, cAMP-specific PDEs (PDE IVs) as potential anti-inflammatory and anti-asthmatic drugs. As mentioned above, the cloning and expression of a cDNA that encodes a human PDE IV subtype expressed in monocytes (hPDE IV.sub.A) has been reported. This enzyme exhibited significant amino acid sequence homology to PDE IVs from rat brain (Colicelli, supra (1989)) and Drosophila (Chen, C-N. et al., Proc. Nat'l. Acad. Sci USA 83:9313-17 (1986)). Furthermore, the recombinant enzyme was overexpressed in both yeast and mammalian cells and defined as a PDE IV based on its kinetic characteristics and sensitivity to isozyme-selective inhibitors. Recombinant hPDE IVA possesses a low K.sub.m for cAMP (K.sub.m =3.2 .mu.M), a high K.sub.m for cGMP, and is inhibited by rolipram (K.sub.i =0.06 .mu.M) but not by selective inhibitors of other PDE isozymes.
It has been proposed that the anti-depressant activity of the PDE IV-selective inhibitor rolipram is associated with the inhibition of PDE IVs in the central nervous system. Rolipram binds with high affinity to rat brain homogenates and it has been assumed that this binding site represents either a catalytic or allosteric site within the PDE IV molecule itself. Accordingly, it has been recently established that recombinant hPDE IVA possesses both catalytic activity and a high affinity (K.sub.d =2 nM) .sup.3 H!-rolipram binding site (Torphy T. J. et al., J. Biol. Chem. 267:1798-1804 (1992)). Although the relationship between this high affinity binding site and the catalytic activity of hPDE IV.sub.A is not clear, it has been proposed that this site may represent either an allosteric site or the catalytic site on one of two distinct catalytic forms of the enzyme. It is of considerable interest to know if an additional PDE IV subtype is expressed in human brain, and if so, whether this subtype has biochemical characteristics similar to hPDE IV.sub.A. Of particular interest is the determination whether the high affinity rolipram binding site exists on PDE IV subtypes in addition to hPDE IV.sub.A.
Although the mechanism of action of rolipram can be assessed biochemically using the available recombinant PDE IV enzymes derived from human monocytes and rat brain, a true pharmacological understanding of how PDE IV activity (as well as cellular cAMP content) regulates neurobiochemical processes is limited by lack of knowledge regarding PDE IV subtypes expressed in the human brain, if any. Accordingly, it is a purpose of this invention to provide isolated cDNA clones encoding PDE IV from human brain and to employ this valuable reagent in a screening protocol for the discovery of subtype-specific PDE IV inhibitors. Disclosed herein is the cloning of a cDNA from a human frontal cortex cDNA library that encodes a unique PDE IV subtype. This enzyme is designated as HPDE IV.sub.B according to the nomenclature of Beavo and Reifsnyder supra (1990). The cDNA product is defined as a type IV PDE based on its comparative amino acid sequence as well as PDE IV-selective inhibitors, the kinetic characteristics and the .sup.3 H!rolipram binding capacity of the recombinant enzyme. Surprisingly, this PDE IV.sub.B subtype exhibits a restricted tissue-type pattern of expression.